1. Field of the Invention
The present invention relates to the novel thermostable N-acylamino acid racemase (NAAAR) from Deinococcus radiodurans NCHU1003, the coding sequence and the preparation thereof. The present invention also relates to an improved process for the preparation of optically active L-amino acid, such as L-homophenylalanine (L-HPA) and the derivatives thereof, from their corresponding N-protected amino acid by using the novel NAAAR combined with L-N-carbamoylase.
2. Background of the Invention
Optically active amino acids, including the L- and D-form amino acids, have been widely used in the industry of food, feedstuff, pharmaceutical synthesis and agricultural chemistry as preparative materials. However, different type of optically active isomers may exhibit totally different biological activities. For instance, some optically active isomer with certain configuration possesses excellent biological activities, but the other isomer does not have such biological activities. When these two isomers are present at the same time, they not only reduce the biological activities but also further inhibit the existing biological activities. For that reason, it is important to find the way for producing enamtiomeric form of amino acid in the pharmaceutical industry.
In the prior art of optically active amino acid preparation, it was performed with chemical synthesis. However, the process of organic synthesis often results in involved environmental pollution and has high cost in isolation and purification. Accordingly, there are lots of limitations in large-scale production. Asymmetric hydrogenation and enzyme-catalyzed reaction were therefore developed for producing high purity optically active amino acids and derivatives thereof. Frank J. Villani Jr et al (1998) isolated the racemic amino acids with tartaric acid and salicylaldehyde, which obtains a product of 32% yield, with e.e. (enantiomeric excess) value of 94%. According to the literatures, there are many bioconversion methods for preparing L-amino acids, but most of such methods still remain nearly 50% of D-formed materials. Based on that the advantages of substrate-specificity, stereochemical selectivity and the like, enzyme-catalyzed process are used in the racemation of amino acid for the production of highly pure optically active amino acids. Non-naturally occurred L-amino acids can be produced by using D,L-5-monosubstituted acetolactams as substrates and Arthrobacter sp. DSM3745 as biocatalyst (Syldatk et al 1992). Similar methods have been applied to other microorganisms, such as Pseudomonas, Achromobacter, Serratia, Aspergillus and so on. Tseng et al (1991) used an enzyme-catalyzed specific stereoselective acetylation of (R)-2-hydroxyl-4-phenylbutyronitrile to produce L-amino acids, but this method required more than 12 days to complete the reaction.
Chen et al (1999) used aspartic acid and 2-oxo-4-phenylbutyric acid as substrates and the enzyme tyrosine aminotransferase to perform an equilibrium shift controlled enzymatic reaction for the production of pure L-HPA. Zhao et al use a commercialized protease—alcalase to produce L-HPA in large-scale in which an e.e. value of 98% can be obtained. This method used N-protected amino acid as substrate, and only L-form N-protected amino acid could be hydrolysed, so that can accomplish the isolation of desired products. However, such enzyme-catalyzed reaction is very complicate, for that the substrate must be protected previously and the protective group should be removed after the enzyme-catalyzed reaction. An alternative enzyme-catalyzed method are preformed by using L-amino acid acylase, which specifically acts on N-protected amino acid, to convert N-protected amino acid racemic mixture to L- or D-formed amino acid (EP99118844 and JP11318442A). Thereafter, the remaining D-formed N-protected amino acid may be converted to L-formed N-protected amino acid by physical or chemical racemation (as described in JP656775A). The difficulty of such process was the reaction must be repeated again and the product and reactants must be isolated after each reaction. Moreover, reaction temperature and pH are increased to racemize the remaining D-formed N-protected amino acid. L-Amino acid may also be produced by using amino acid acyl transferase, which specifically acts on the N-protected amino acid, and further racemation to convert remaining D-formed N-protected amino acid to L-form amino acid (EP99118844.2; and Bommarius A. S. et al, Tetrahedron Asymmetry, 1997, 8:3197-3200).
The N-acylamino acid racemase from actinomycetes was firstly discovered by Takahashi (1991). This enzyme only performs racemation on N-protected amino acids and not on non-protected amino acids. Only few NAAARs were reported to which most of them are from actinomycetes, such as Streptomyces atratus Y-53 (Tokuyama et al, Appl. Microbiol. Biotechnol. 1994, 40: 835-840; and Amycolatopis sp. TS-1-60, Tokuyama et al, Appl. Microbiol. Biotechnol. 1995a, 42: 853-859). The NAAAR from Amycolatopis sp. TS-1-60 can maintains its activity at 55° C. for about 30 min, but the presence of D, L-amino acid acyl transferase in Amycolatopis can interfere the optical purity of methionine, thus limits its application.
L-homophenylalanine (L-HPA) is an optically active unnatured amino acid which can be useful as intermediates for the synthesis of many antihypertension drugs, ACE inhibitors. So far the high purity of L-HPA and derivative thereof is produced by chemical resolution or asymmetric hydrogenation (U.S. Pat. No. 5,981,794 and EP00902011A1). However, the previous methods must be carried out in a condition of high temperature and strong base, which exhibits the problems in expense, safety and environmental conservation and could not achieve best ee value and yield. The process of present invention is an enzyme catalytic method, so its reaction condition may avoid the disadvantages occurred in the chemical methods.
The production of L-HPA by enzymetic processes has been focused on amino transferase using L-aspartic acid or L-glutamic acid as the amino donor, and 2-oxo-4-phenyl butyric acid as the amino acceptor to obtain high purity L-PHA. Syldatk, C et al (1992) used the hydantoinase capable of catalying D, L-5-monosubstituted hydantoins in combination with D-N-carbamoylase or L-N-carbamoylase to produce novel D- or L-formed amino acids. The hydantoinase process had been applied in the production of L-PHA (Lo & Hsu 2003). In this process the hydantoinase is isolated from B. agri, since the substrate specificity of this hydantoinase is favor D-form homophenylalaninyl hydantoin (D-HPAH), a large amount of intermediate (D-NCaHPA) is formed during the process and therefore disadvantaged to the biosynthesis of L-HPA.
The present invention provides an enzymetic process for producing L-HPA with high optical purity by using a novel thermostable N-acylamino acid racemase (NAAAR) from Deinococcus radiodurans NCHU1003 and a carbamoylase from Deinococcus sp.